Hepatitis C virus is an important human pathogen that can cause severe liver diseases including liver cirrhosis and liver cancer. This virus has a positive-stranded RNA genome that encodes a polyprotein, which is cleaved by cellular and viral proteases to generate ten mature protein products. Recent research indicates that, in addition to this polyprotein, HCV also encodes a 17 kDa protein using an alternative reading frame. This protein, which is named the F (or ARF) protein, is synthesized by ribosomal frameshift during translation. The goal of this application is to continue our research to understand the biological functions of the HCV F protein and the molecular mechanism that regulates its expression. There are three specific aims in this research application. The focus of Aim 1 is to study the molecular mechanism that regulates the HCV ribosomal frameshift for the synthesis of the F protein. The HCV ribosomal frameshift signal is A-rich and followed by a sequence that can form a double stem-loop structure. The goal of this aim is to investigate how this signal mediates translational frameshift and the possible roles of the double stem-loop structure and other viral factors in the regulation of this frameshifting process. The HCV F protein is a phosphoprotein that is associated with membranes. The focus of Aim 2 is to investigate the membrane topology of the F protein and how this protein is phosphorylated. The purpose of this aim is to understand how these biological characteristics of the F protein play a role in the HCV life cycle. The F protein is expressed during natural infection and is conserved in the great majority of the HCV sequences reported in the database. These observations as well as the finding of a similar gene in the genome of the related GB virus type B suggest an important role of this protein in the HCV life cycle. The focus of Aim 3 is to study the possible functions of the F protein in the HCV life cycle.